Maintenance of R-loop structures by phosphorylated hTERT preserves genome integrity.

As aberrant accumulation of RNA-DNA hybrids (R-loops) causes DNA damage and genome instability, cells express regulators of R-loop structures. Here we report that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates R-loop formation. We found that the phosphorylated form of hTERT (p-hTERT) exhibits RdRP activity in nuclear speckles both in telomerase-positive cells and telomerase-negative cells with alternative lengthening of telomeres (ALT) activity. The p-hTERT did not associate with telomerase RNA component in nuclear speckles but, instead, with TERRA RNAs to resolve R-loops. Targeting of the TERT gene in ALT cells ablated RdRP activity and impaired tumour growth. Using a genome-scale CRISPR loss-of-function screen, we identified Fanconi anaemia/BRCA genes as synthetic lethal partners of hTERT RdRP. Inactivation of RdRP and Fanconi anaemia/BRCA genes caused accumulation of R-loop structures and DNA damage. These findings indicate that RdRP activity of p-hTERT guards against genome instability by removing R-loop structures.

Endoplasmic reticulum transporter OAT2 regulates drug metabolism and interaction.

Xenobiotic metabolic reactions in the hepatocyte endoplasmic reticulum (ER) including UDP-glucuronosyltransferase and carboxylesterase play central roles in the detoxification of medical agents with small- and medium-sized molecules. Although the catalytic sites of these enzymes exist inside of ER, the molecular mechanism for membrane permeation in the ER remains enigmatic. Here, we investigated that organic anion transporter 2 (OAT2) regulates the detoxification reactions of xenobiotic agents including anti-cancer capecitabine and antiviral zidovudine, via the permeation process across the ER membrane in the liver. Pharmacokinetic studies in patients with colorectal cancer revealed that the half-lives of capecitabine in rs2270860 (1324C > T) variants was 1.4 times higher than that in the C/C variants. Moreover, the hydrolysis of capecitabine to 5'-deoxy-5-fluorocytidine in primary cultured human hepatocytes was reduced by OAT2 inhibitor ketoprofen, whereas capecitabine hydrolysis directly assessed in human liver microsomes were not affected. The immunostaining of OAT2 was merged with ER marker calnexin in human liver periportal zone. These results suggested that OAT2 is involved in distribution of capecitabine into ER. Furthermore, we clarified that OAT2 plays an essential role in drug-drug interactions between zidovudine and valproic acid, leading to the alteration in zidovudine exposure to the body. Our findings contribute to mechanistically understanding medical agent detoxification, shedding light on the ER membrane permeation process as xenobiotic metabolic machinery to improve chemical changes in hydrophilic compounds.

Association of VEGFA and CCL4L2 polymorphisms with hand-foot skin reaction and survival of regorafenib in Japanese patients with colorectal cancer.

PURPOSE: Treatment with regorafenib, which inhibits vascular endothelial growth factor (VEGF) receptor, frequently results in hand-foot skin reaction (HFSR), requiring treatment discontinuation or dose reduction. In our prospective study of regorafenib on patients with metastatic colorectal cancer, 17% of patients developed grade 3 HFSR. Herein, we retrospectively examined genetic polymorphisms associated with regorafenib-induced severe HFSR. METHODS: To identify associated polymorphisms, exploratory whole-exome sequencing focusing on factors related to VEGF-mediated signaling pathways was first performed in seven patients each, with grade 3 HFSR and without HFSR. The identified HFSR-associated polymorphisms were analyzed in all the 40 patients. RESULTS: The genotype frequency of rs3025009 G/A or A/A in the gene encoding VEGF-A (VEGFA) in patients with ≥ grade 2 HFSR was significantly higher than in other patients (P = 0.0257, Pc = 0.0771 [Bonferroni correction]). The frequency of C-C motif of chemokine ligand 4-like 2 (CCL4L2) rs3744596 A/T or T/T in patients with grade 3 HFSR was significantly lower than in others (P = 0.00894, Pc = 0.0268). The combination of the risk genotypes VEGFA rs3025009 G/A or A/A and CCL4L2 rs3744596 A/A was significantly associated with a higher incidence of grade 3 (P = 0.000614, Pc = 0.00246) and a longer median progression-free survival (P = 0.0234) than others. CONCLUSIONS: These VEGF-related polymorphisms were found to be associated with HFSR and the survival benefits of regorafenib treatment. TRIAL REGISTRATION NUMBER AND DATE: UMIN000013939, registered on May 12, 2014, when 6 months after the approval by the Institutional Review Board of Showa University.

Persimmon tannin promotes the growth of Saccharomyces cerevisiae under ethanol stress.

BACKGROUND: Saccharomyces cerevisiae plays a pivotal role in various industrial processes, including bioethanol production and alcoholic beverage fermentation. However, during these fermentations, yeasts are subjected to various environmental stresses, such as ethanol stress, which hinder cell growth and ethanol production. Genetic manipulations and the addition of natural ingredients rich in antioxidants to the culture have been shown to overcome this. Here, we investigated the potential of persimmon tannins, known for their antioxidative properties, to enhance the ethanol stress tolerance of yeast. RESULTS: Assessment of the effects of 6.25 mg mL-1 persimmon tannins after 48 h incubation revealed cell viability to be increased by 8.9- and 6.5-fold compared to the control treatment with and without 12.5% ethanol, respectively. Furthermore, persimmon tannins reduced ethanol-induced oxidative stress, including the production of cellular reactive oxygen species and acceleration of lipid peroxidation. However, persimmon tannins could hardly overcome ethanol-induced cell membrane damage. CONCLUSION: The findings herein indicate the potential of persimmon tannin as a protective agent for increasing yeast tolerance to ethanol stress by restricting oxidative damage but not membrane damage. Overall, this study unveils the implications of persimmon tannins for industries relying on yeast. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Terminal regions of UAP56 and URH49 are required for their distinct complex formation functioning to an essential role in mRNA processing and export.

UAP56 and URH49 are closely related RNA helicases that function in selective mRNA processing and export pathways to fine-tune gene expression through distinct complex formations. The complex formation of UAP56 and URH49 is believed to play a crucial role in regulating target mRNAs. However, the mechanisms underlying this complex formation have not been fully elucidated. Here we identified the regions essential for the complex formation of both helicases. The terminal regions of UAP56 and the C-terminal region of URH49 were indispensable for their respective complex formation. Further analysis revealed that a specific amino acid at the C-terminus of UAP56 is critical for its complex formation. Alanine substitution of this amino acid impairs its complex formation and subsequently affected its mRNA processing and export activity. Our study provides a deeper understanding of the basis for the complex formation between UAP56 and URH49.

Structural differences between the closely related RNA helicases, UAP56 and URH49, fashion distinct functional apo-complexes.

mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2.

Vincristine Disposition and Neurotoxicity Are Unchanged in Humanized CYP3A5 Mice.

Previous studies have suggested that the incidence of vincristine-induced peripheral neuropathy (VIPN) is potentially linked with cytochrome P450 (CYP)3A5, a polymorphic enzyme that metabolizes vincristine in vitro, and with concurrent use of azole antifungals such as ketoconazole. The assumed mechanism for these interactions is through modulation of CYP3A-mediated metabolism, leading to decreased vincristine clearance and increased susceptibility to VIPN. Given the controversy surrounding the contribution of these mechanisms, we directly tested these hypotheses in genetically engineered mouse models with a deficiency of the entire murine Cyp3a locus [Cyp3a(-/-) mice] and in humanized transgenic animals with hepatic expression of functional and nonfunctional human CYP3A5 variants. Compared with wild-type mice, the systemic exposure to vincristine was increased by only 1.15-fold (95% confidence interval, 0.84-1.58) in Cyp3a(-/-) mice, suggesting that the clearance of vincristine in mice is largely independent of hepatic Cyp3a function. In line with these observations, we found that Cyp3a deficiency or pretreatment with the CYP3A inhibitors ketoconazole or nilotinib did not influence the severity and time course of VIPN and that exposure to vincristine was not substantially altered in humanized CYP3A5*3 mice or humanized CYP3A5*1 mice compared with Cyp3a(-/-) mice. Our study suggests that the contribution of CYP3A5-mediated metabolism to vincristine elimination and the associated drug-drug interaction potential is limited and that plasma levels of vincristine are unlikely to be strongly predictive of VIPN. SIGNIFICANCE STATEMENT: The current study suggests that CYP3A5 genotype status does not substantially influence vincristine disposition and neurotoxicity in translationally relevant murine models. These findings raise concerns about the causality of previously reported relationships between variant CYP3A5 genotypes or concomitant azole use with the incidence of vincristine neurotoxicity.

A polymorphism in ABCA2 is associated with neutropenia induced by capecitabine in Japanese patients with colorectal cancer.

PURPOSE: Capecitabine is a prodrug that converts to 5-fluorouracil (5-FU) in three steps. A previous study showed that ABCA2 rs2271862 (C > T) and ABCG5 rs6720173 were associated with increased clearance of 5-FU and 5'-deoxy-5-fluorouridine, respectively, in Spanish patients with colorectal cancer (CRC) (Br J Clin Pharmacol 2021) and reported that ABCA2 rs2271862 was associated with decreased risk of capecitabine-induced neutropenia. Other studies have reported that ABCB1 rs1128503, rs2032592, and rs1045642 were associated with capecitabine-induced toxicity in Spanish CRC patients (Oncotarget 2015, Phamacogenomics 2010). Here, we prospectively examined the effects of ABC transporter genes polymorphisms on capecitabine pharmacokinetics and toxicity. METHODS: We enrolled patients with postoperative CRC treated with adjuvant capecitabine plus oxaliplatin (CapeOX) and patients with metastatic CRC receiving CapeOX. Pharmacokinetic analysis of the first capecitabine dose (1000 mg/m2) was performed on day 1. We analyzed plasma concentrations of capecitabine and its three metabolites by high-performance liquid chromatography and ABC transporter genes polymorphisms using direct sequencing. RESULTS: Patients with ABCA2 rs2271862 T/T genotype had significantly lower area under the plasma concentration-time curve of capecitabine, but not of its metabolites, which were divided by the dose of the parent drug, than patients with C/C or C/T genotype (P = 0.0238). Frequency of ≥ grade 2 neutropenia was significantly lower in patients with ABCA2 rs2271862 T/T genotype (P = 0.00915). Polymorphisms in ABCG5 and ABCB1 were not associated with capecitabine pharmacokinetics and toxicity. CONCLUSIONS: We found that ABCA2 polymorphism was significantly associated with systemic exposure to capecitabine and capecitabine-induced neutropenia in Japanese patients with CRC.

Associations of HLA-C*01:02 and HLA-B*46:01 with regorafenib-induced erythema multiforme in Japanese patients with metastatic colorectal cancer.

Regorafenib improves the survival of patients with metastatic colorectal cancer (mCRC); however, it is also characterized by detrimental dermal side effects that may require treatment cessation or modified dosing. In our previous prospective pharmacokinetic, pharmacodynamic, and pharmacogenetic studies, 17.5% (7/40) of the patients with mCRC had grade 3 erythema multiforme (EM) that caused treatment discontinuation. Haplotypes in genes encoding human leukocyte antigen (HLA) are associated with EM following the administration of drugs, such as allopurinol. This study examined the association between HLA haplotypes and regorafenib-induced EM. Regorafenib was administered orally at 160 mg/body once daily for weeks 1-3 of each 4-week cycle. To determine the HLA haplotypes, we used the WAKFlow HLA Typing Kit HLA-A, -B, or -C. The carrier frequency of HLA-C*01:02 in patients with EM (6/7) was higher than that in tolerant controls (8/33; odds ratio [OR] = 18.8, 95% confidence interval [CI] = 1.95-180, p = 0.00437). HLA-B*46:01 was also associated with EM (OR = 11.6, 95% CI = 1.47-92.1, p = 0.0299). These associations were no longer significant after Bonferroni correction for multiple testing. Therefore, regorafenib-induced EM in Japanese patients appears to be associated with specific HLA haplotypes but further validation is needed.

Dehydrozingerone enhances the fungicidal activity of glabridin against Saccharomyces cerevisiae and Candida albicans.

Drug resistance commonly occurs when treating immunocompromized patients with fungal infections. Dehydrozingerone-a phenolic compound isolated from the rhizome of Zingiber officinale-inhibits drug efflux in Saccharomyces cerevisiae by overexpression of the ATP-binding cassette (ABC) transporter Pdr5p. We aimed to investigate whether dehydrozingerone enhances the antifungal activity of glabridin-an isoflavan isolated from the roots of Glycyrrhiza glabra L.-by attenuating multidrug resistance through the intrinsic expression system of multidrug-efflux-related genes in a wild-type strain of the model yeast. The antifungal activity of 50 µmol l-1 glabridin alone was weak and temporary against S. cerevisiae; however, cell viability was significantly inhibited when the cells were co-treated with glabridin and dehydrozingerone. This enhancement was also observed in human pathogenic Candida albicans. Glabridin efflux did not depend on a particular drug efflux pump; instead, the transcription factors PDR1 and PDR3-regulating the transcription of multiple genes encoding drug efflux pumps-were involved in the antifungal activity and efflux of glabridin. qRT-PCR analysis revealed that dehydrozingerone reduced glabridin-induced overexpression of the ABC transporter-related genes PDR1, PDR3, and PDR5 to the levels observed in untreated cells. Our findings indicated that dehydrozingerone potentiates the efficacy of plant-derived antifungals through its effects on ABC transporters.

Adeno-associated virus-mediated knockdown demonstrates the major role of hepatic Bcrp in the overall disposition of the active metabolite of the tyrosine kinase inhibitor regorafenib in mice.

Breast cancer resistance protein (BCRP) is expressed on hepatic bile canalicular membranes; however, its impact on substrate drug disposition is limited. This study proposes an in vivo knockdown approach using adeno-associated virus encoding short hairpin RNA (shRNA) targeting the bcrp gene (AAV-shBcrp) to clarify the substrate, the overall disposition of which is largely governed by hepatic Bcrp. The disposition of the tyrosine kinase inhibitor, regorafenib, was first examined in bcrp gene knockout (Bcrp-/-) and wild-type (WT) mice, as it was sequentially converted to active metabolites M - 2 and M - 5, which are BCRP substrates. After oral administration of regorafenib, plasma and liver concentrations of M - 5, but not regorafenib, were higher in Bcrp-/- than WT mice. To directly examine the role of hepatic Bcrp in M - 5 disposition, M - 5 was intravenously injected into mice three weeks after the intravenous injection of AAV-shBcrp, when mRNA of Bcrp in the liver (but not the small intestine) was downregulated. AAV-shBcrp-treated mice showed higher M - 5 concentration in plasma and liver, but lower biliary excretion than the control mice, indicating the fundamental role of hepatic Bcrp in M - 5 disposition. This is the first application of AAV-knockdown strategy to clarify the pharmacokinetic role of xenobiotic efflux transporters in the liver.

Pharmacokinetics of gefitinib in elderly patients with EGFR-mutated advanced non-small cell lung cancer: a prospective study.

BACKGROUND: Gefitinib is recommended as a first-line treatment option for elderly patients with non-small cell lung cancer (NSCLC). Because no pharmacokinetics of gefitinib have been examined, we prospectively assessed the pharmacokinetics of gefitinib in patients with epidermal growth factor receptor gene-mutated advanced NSCLC who were 75 years or older. METHODS: Gefitinib was orally administered once daily at a dose of 250 mg. The concentrations of gefitinib and its major metabolite O-desmethyl gefitinib in plasma were measured by high-performance liquid chromatography. The area under the plasma concentration-time curve from time 0 to 48 h (AUC0-48) was calculated. Polymorphisms in CYP3A5, CYP2D6, ABCG2, ABCB1, and OATP1B1 were analyzed by direct sequencing. RESULTS: Eighteen patients with a median age of 80.5 years (range, 75-89) with adequate liver and kidney functions were examined. AUC0-48 values of gefitinib and O-desmethyl gefitinib in this population were 9.49 ± 3.5 and 10.6 ± 14 µM h, respectively. Compared to the gefitinib pharmacokinetics observed in a previous phase I study in Japan, systemic exposure to gefitinib in elderly patients was slightly higher than that in younger patients. Three patients experienced grade 3 diarrhea, increases in alanine aminotransferase, and aspartate aminotransferase levels 30 days after starting gefitinib treatment. The CYP2D6 genotype was associated with CYP2D6-mediated metabolism of gefitinib to O-desmethyl gefitinib. CONCLUSIONS: We demonstrated for the first time the systemic exposure to gefitinib in elderly patients with NSCLC. TRIAL REGISTRATION: The study was registered with the University Hospital Medical Information Network-Clinical Trials Registry Japan (UMIN000026409) on November 8, 2013.

MPP6 stimulates both RRP6 and DIS3 to degrade a specified subset of MTR4-sensitive substrates in the human nucleus.

Recent in vitro reconstitution analyses have proven that the physical interaction between the exosome core and MTR4 helicase, which promotes the exosome activity, is maintained by either MPP6 or RRP6. However, knowledge regarding the function of MPP6 with respect to in vivo exosome activity remains scarce. Here, we demonstrate a facilitative function of MPP6 that composes a specific part of MTR4-dependent substrate decay by the human exosome. Using RNA polymerase II-transcribed poly(A)+ substrate accumulation as an indicator of a perturbed exosome, we found functional redundancy between RRP6 and MPP6 in the decay of these poly(A)+ transcripts. MTR4 binding to the exosome core via MPP6 was essential for MPP6 to exert its redundancy with RRP6. However, at least for the decay of our identified exosome substrates, MTR4 recruitment by MPP6 was not functionally equivalent to recruitment by RRP6. Genome-wide classification of substrates based on their sensitivity to each exosome component revealed that MPP6 deals with a specific range of substrates and highlights the importance of MTR4 for their decay. Considering recent findings of competitive binding to the exosome between auxiliary complexes, our results suggest that the MPP6-incorporated MTR4-exosome complex is one of the multiple alternative complexes rather than the prevailing one.

Application of Personal Health Record in Enhancing the Quality of Life in Patients With Breast Cancer Who Received Adjuvant Hormonal Therapy.

Objective: Recently, personal health records (PHR) have become a communication tool between patients and medical professionals. PHR applications (PHR app) can be installed on smartphones to record patient-reported outcomes (PROs). This study prospectively examined whether patients with breast cancer could record PROs, including subjective and objective symptoms, on PHR app. Materials and Methods: Patients who received adjuvant hormonal therapy were enrolled. The patients were asked to collect PROs related to physical conditions, symptoms, and medications on their PHR app from the beginning of therapy for one month. Quality of life (QoL) was evaluated before treatment initiation and one month after. Patients completed a questionnaire of their opinions concerning the PHR app after use. Results: Fourteen patients were enrolled between October and December 2020. All patients could use the PHR app during the study period without any negative effects on QoL. Eleven (79%) patients fully recorded their PROs on the app. Typical side effects induced by hormonal therapy to reduce the QoL were observed (hot flash in two patients, 14.3%). The questionnaire revealed that approximately 70% wanted to use the PHR app in the future to communicate with medical staff and to report adverse events. Specifically, 90% of patients who experienced difficulty communicating with medical staff wanted to use the PHR app. Some patients wanted to utilize the PHR app to set reminders to take medications. Conclusion: The PHR app can be applied as a communication tool between patients taking adjuvant hormonal therapy and medical professionals.

Baseline soluble MICA levels act as a predictive biomarker for the efficacy of regorafenib treatment in colorectal cancer.

BACKGROUND: To evaluate the effect of regorafenib on soluble MHC class I polypeptide-related sequence A (MICA) (sMICA) level in vitro. In addition, we clinically examined whether its plasma levels were associated with regorafenib activity in terms of progression-free survival (PFS) in patients with CRC. METHODS: Human CRC cell line HCT116 and HT29 cells were treated with regorafenib and its pharmacologically active metabolites, M2 or M5 at the same concentrations as those in sera of patients. We also examined the sMICA levels and the area under the plasma concentration-time curve of regorafenib, M2 and M5. RESULTS: Regorafenib, M2, and M5 significantly suppressed shedding of MICA in human CRC cells without toxicity. This resulted in the reduced production of sMICA. In the clinical examination, patients with CRC who showed long median PFS (3.7 months) had significantly lower sMICA levels than those with shorter median PFS (1.2 months) (p = 0.045). CONCLUSIONS: MICA is an attractive agent for manipulating the immunological control of CRC and baseline sMICA levels could be a predictive biomarker for the efficacy of regorafenib treatment.

CHERP Regulates the Alternative Splicing of pre-mRNAs in the Nucleus.

Calcium homeostasis endoplasmic reticulum protein (CHERP) is colocalized with the inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum or perinuclear region, and has been involved in intracellular calcium signaling. Structurally, CHERP carries the nuclear localization signal and arginine/serine-dipeptide repeats, like domain, and interacts with the spliceosome. However, the exact function of CHERP in the nucleus remains unknown. Here, we showed that poly(A)+ RNAs accumulated in the nucleus of CHERP-depleted U2OS cells. Our global analysis revealed that CHERP regulated alternative mRNA splicing events by interaction with U2 small nuclear ribonucleoproteins (U2 snRNPs) and U2 snRNP-related proteins. Among the five alternative splicing patterns analyzed, intron retention was the most frequently observed event. This was in accordance with the accumulation of poly(A)+ RNAs in the nucleus. Furthermore, intron retention and cassette exon choices were influenced by the strength of the 5' or 3' splice site, the branch point site, GC content, and intron length. In addition, CHERP depletion induced anomalies in the cell cycle progression into the M phase, and abnormal cell division. These results suggested that CHERP is involved in the regulation of alternative splicing.

Discrimination of hand-foot skin reaction caused by tyrosine kinase inhibitors based on direct keratinocyte toxicity and vascular endothelial growth factor receptor-2 inhibition.

Tyrosine kinase inhibitors (TKIs) are molecular-targeted anticancer drugs. Their benefits are limited by dermal toxicities, including hand-foot skin reaction (HFSR), which is commonly found in skin areas subjected to friction. The present study aimed to explain the incidence of HFSR in patients treated with TKIs by focusing on keratinocyte toxicity and inhibition of vascular endothelial growth factor receptor (VEGFR), which plays an essential role in angiogenesis. Mice with gene knockout for the immunosuppressive cytokine interleukin-10 exhibited HFSR-like phenotypes, such as cytotoxicity in keratinocytes and increased number and size of blood vessels after repeated doses of regorafenib, sorafenib, and pazopanib, all of which cause high incidence of HFSR, in combination with tape-stripping mimicking skin damage at the friction site. Comprehensive examination of the direct cytotoxic effects of 21 TKIs on primary cultured human keratinocytes revealed that 18 of them reduced the cell viability dose-dependently. Importantly, the ratio of the trough concentration in patients (Ctrough) to the LC50 values of cell viability reduction was higher than unity for four HFSR-inducing TKIs, suggesting that these TKIs cause keratinocyte toxicity at clinically relevant concentrations. In addition, eight HFSR-inducing TKIs caused inhibition of VEGFR-2 kinase activity, which was validated by their ratios of Ctrough to the obtained IC50,VEGFR-2 of more than unity. All 12 TKIs with no reported incidence of HFSR exhibited less than unity values for both Ctrough/LC50,keratinocytes and Ctrough/IC50,VEGFR-2. These results suggested that a combination of keratinocyte toxicity and VEGFR-2 inhibition may explain the incidence of HFSR upon TKI usage in humans.

Carboxylative Cyclization of a Propargylic Amine with CO2 Catalyzed by a Silica-Coated Magnetite.

By employing a silica-coated magnetite as a catalyst, a silica-catalyzed carboxylative cyclization of propargylic amines with carbon dioxide (CO2) proceeded to afford the corresponding 2-oxazolidinones. Moreover, after the reaction, the silica-coated magnetic catalyst was readily recovered by use of an external magnet and could be reused up to six times without deactivation.

Characteristics of miRNA-SNPs in healthy Japanese subjects and non-small cell lung cancer, colorectal cancer, and soft tissue sarcoma patients.

Single nucleotide polymorphisms in genes encoding microRNAs (miRNA-SNPs) may affect the maturation steps of miRNAs or target mRNA recognition, leading to changes in the expression of target mRNAs to cause gain- or loss-of-function changes. Several miRNA-SNPs are known to be associated with the risk of diseases such as cancer. The purpose of this study was to comprehensively determine the miRNA-SNPs in Japanese individuals to evaluate the differences in allele frequencies between ethnicities by comparing data from the global population in the 1000 Genomes Project and differences between healthy subjects and cancer patients. We performed next-generation sequencing targeting genes encoding 1809 pre-miRNAs. As a result, 403 miRNA-SNPs (146 miRNA-SNPs per subject on average) were identified in 28 healthy Japanese subjects. We observed significant differences in the allele frequencies between ethnicities in 33 of the 403 miRNA-SNPs. The numbers of miRNA-SNPs per subject in 44 non-small cell lung cancer (NSCLC), 33 colorectal cancer (CRC), and 15 soft tissue sarcoma (STS) patients were almost equal to those in healthy subjects. Significant differences in allele frequencies were observed for 14, 11, and 9 miRNA-SNPs in NSCLC, CRC, and STS patients compared with the frequencies in healthy subjects, suggesting that these SNPs can be biomarkers of risk for each type of cancer assessed. In summary, we comprehensively characterized miRNA-SNPs in Japanese individuals and found differences in allele frequencies of several miRNA-SNPs between ethnicities and between healthy subjects and cancer patients. Studies investigating a larger number of subjects should be performed to confirm the potential of miRNA-SNPs as biomarkers of cancer risk.

The Exon Junction Complex Core Represses Cancer-Specific Mature mRNA Re-splicing: A Potential Key Role in Terminating Splicing.

Using TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that mRNA re-splicing is controlled by specific repressors, and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH, or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.